In general, reporter constructs rely upon natural mechanisms for sensing an extracellular ligand via a reporter, inducing an intracellular signaling cascade (such as a relay of kinases), and eventually causing a transcription factor to be active and present in the cell nucleus. The reporter construct may incorporate a natural promoter (which may be regulated by multiple transcription factors) or an engineered promoter (which may be regulated by one specific transcription factor) to drive the conditional expression of a reporter gene. While this approach is useful for monitoring native signaling, it is not well-suited to robust biosensor engineering for several reasons. Because native receptors and signaling proteins are required, these components must either be already present in the cell type of interest or they must be exogenously expressed (e.g., by transfection of expression plasmids) at levels that guarantee adequate ligand-inducible signaling without risking ligand-independent constitutive signaling. In addition, native mechanisms often exist for promoting or suppressing the activity of receptors, intracellular signaling proteins, and promoters. Consequently, it is often not efficient (or even possible) to transfer reporter systems between cell types, and potential interference by native regulatory mechanisms complicates the interpretation of reporter gene outputs.
The TANGO assay system is marketed by Life Technologies and was originally described by Barnea et al. (Proc Natl Acad Sci USA. 2008 Jan. 8; 105(1):64-9). The TANGO system is designed to detect the interaction of two native proteins: one protein is fused (genetically) to the Tev protease and the second protein is fused (genetically) to a Tev PCS-transcription factor domain. In TANGO, when the two engineered proteins are held in proximity, which is mediated by a native protein-protein interaction, Tev cleaves the PCS to release the transcription factor. Although the TANGO approach can be used to monitor signaling through a native receptor, this strategy still relies upon native mechanisms and interactions (such as ligand binding-dependent recruitment of an intracellular signaling protein to a receptor complex), which are subject to interference from native regulatory mechanisms. Moreover, designing a new TANGO biosensor requires identifying both a suitable native receptor and a corresponding intracellular signaling protein or proteins.